Parkinson's disease is a common neurodegenerative motor disorder caused by dopaminergic neuron death. Parkinson's is characterized by the presence of Lewy bodies, intracellular plaques primarily composed of alpha synuclein. While large insoluble alpha synuclein aggregates are found in Lewy bodies, smaller soluble alpha synuclein oligomers which can permeabilize membranes are implicated as a neuron-killing species in Parkinson's. Alpha synuclein is normally a natively unfolded, monomeric protein abundantly expressed in the brain. The goal of this project is to understand the conversion of aS from a natively unfolded monomer to a toxic oligomeric species. Single molecule fluorescence methods will be used to probe the changes in alpha synuclein conformational states that underpin this transition. A better understanding of these conformational states and the associated changes in alpha synuclein will identify therapeutic targets to treat PD at the earliest possible stages. The first aim of this project will use single molecule Forster resonance energy transfer (smFRET) to investigate partial folding in monomeric alpha synuclein that precedes oligomerization. This will determine which regions of alpha synuclein undergo the most relevant conformational changes and what solution conditions cause these changes. The second aim of this project seeks to purify and study disease-relevant oligomeric alpha synuclein species. smFRET will also be used in this aim in order to correlate certain conformational states in oligomers with their cytotoxicity. Finally, the third aim of this project is to measure alpha synuclein conformational state within cells. Correlating in vitro biophysical characteristics of alpha synuclein with intracellular states will establish which conformational states studied in Aims 1 and 2 are most relevant to aS disease pathways. Total internal reflection fluorescence microscopy will be used to image alpha synuclein within cells to establish localization and to make FRET measurements as well. These aims will characterize the conformational states of alpha synuclein at each step from unfolded monomer to toxic oligomer. Knowledge of these conformational states should inform a search for therapeutic compounds to inhibit oligomerization or alleviate toxicity in order to treat Parkinson's disease. PUBLIC HEALTH RELEVANCE: This project seeks to understand some of the earliest events leading to neuron death and Parkinson's disease pathology. By studying toxic states of the protein alpha synuclein, we will identify targets for treatment strategies designed to prevent the formation of these toxic structures or alleviate the symptoms they cause.